Reduction in minipubertal gonadotropin levels alters reproductive lifespan and ovarian follicular loss in female mice.

study questionWhat is the effect of attenuating the physiological hypergonadotropic activity encountered at minipuberty on female reproductive function in a mouse model? SUMMARY ANSWER: Decreasing the surge of gonadotropins at minipuberty extended reproductive lifespan, coinciding with alterations in neuroendocrine and ovarian aging. WHAT IS KNOWN ALREADY: Minipuberty is characterized by the tremendous activation of the gonadotrope axis, as evidenced by elevated levels of gonadotropins regulating folliculogenesis and the synthesis of ovarian hormones, but its role in fertility remains unclear. STUDY DESIGN, SIZE, DURATION: To determine the link between gonadotrope axis activity at minipuberty and reproductive parameters, we used a pharmacological approach to suppress gonadotropin levels in Swiss mice by injecting daily a GnRH receptor antagonist (GnRHR) (Ganirelix, 10 µg/mouse) or its vehicle between 10 and 16 postnatal days, to cover the entire duration of minipuberty. We analyzed the onset of puberty and estrous cyclicity as well as fertility in young (3-5 months) and middle-aged (11 months) mice from control (CTR) and antagonist-treated groups (n = 17-20 mice/age and treatment group). Ovaries and brains were collected, fixed, and sectioned (for histology, follicle count, and immunohistochemistry) or frozen (for analysis of follicular markers, aging, and inflammation) from adult females, and blood was collected by cardiac puncture for hormonal assays (n = 3-8 mice/age and treatment group). PARTICIPANTS/MATERIALS, SETTING,

methodsTo analyze the initiation of puberty, we monitored vaginal opening and performed vaginal smears in CTR and antagonist-treated mice. We studied estrous cyclicity on vaginal smears at the beginning of reproductive life. Mice were mated several times with males to assess fertility rates, delay of conception, and litter size. To evaluate ovarian function, we counted follicles at different stages and corpora lutea, and we determined the relative intra-ovarian abundance of key follicular markers by real-time RT-PCR, as well as the levels of circulating anti-Müllerian hormone (AMH) and progesterone by ELISA and GC-MS, respectively. We also analyzed features of ovarian aging and inflammation by histology and by measuring the relative intra-ovarian abundance of some markers using real-time RT-PCR. To determine the impact on neuroendocrine determinants related to the CTR of reproduction, we analyzed circulating gonadotropin levels using Luminex assays as well as kisspeptin and GnRH immunoreactivity in the hypothalamus by immunohistochemistry. MAIN RESULTS AND THE ROLE OF CHANCE: Our results show that the treatment had no impact on the initiation of puberty, estrous cyclicity, or fertility at the beginning of reproductive life. However, it increased reproductive lifespan, as shown by the higher percentage of antagonist-treated females than CTRs still fertile at 11 months of age (33% versus 6%; P = 0.0471). There were no significant differences in the number of kisspeptin and GnRH neurons, nor in the density of kisspeptin- and GnRH-immunoreactive neurons in the hypothalamic areas involved in reproduction between the two groups of mice studied at either 4 or 11 months. In addition, basal levels of FSH were comparable between the two groups at 4 and 11 months, but not those of LH at 11 months which were much lower in females treated with antagonist than in their age-matched CTRs (237 ± 59.6 pg/ml in antagonist-treated females versus 1027 ± 226.3 pg/ml in CTRs, P = 0.0069). Importantly, at this age, antagonist-treated mice had basal LH levels comparable to young mice (e.g. in 4-month-old CTRs: 294 ± 71.75 pg/ml, P > 0.05). Despite their prolonged reproductive lifespan and delayed neuroendocrine aging, antagonist-treated mice exhibited earlier depletion of their follicles, as shown by lower numbers of primordial, primary, and preantral follicles associated with lower circulating AMH levels and relative intra-ovarian abundance of Amh transcripts than CTR mice. However, they exhibited comparable completion of folliculogenesis, as suggested by the numbers of antral follicles and corpora lutea, relative intra-ovarian abundance of Cyp19a1, Inhba, and Inhbb transcripts, and circulating progesterone levels that all remained similar to those of the CTR group. These observed alterations in ovarian function were not associated with increased ovarian aging or inflammation. LARGE-SCALE DATA: None. LIMITATIONS, REASONS FOR CAUTION: This study was carried out on mice, which is a validated research model. However, human research is needed for further validation. WIDER IMPLICATIONS OF THE

findingsThis study, which is the first to investigate the physiological role of minipuberty on reproductive parameters, supports the idea that suppressing the high postnatal levels of gonadotropins may have long-term effects on female fertility by extending the duration of reproductive life. Perturbations in gonadotropin levels during this period of life, such as those observed in infants born prematurely, may thus have profound consequences on late reproductive functions. STUDY FUNDING/COMPETING INTEREST(S): This research was conducted with the financial support of ANR AAPG2020 (ReproFUN), CNRS, Inserm, Université Paris Cité, and Sorbonne Université. The authors declare that they have no conflicts of interest.